In situ fluorometer

ABSTRACT

Disclosed herein is a self-contained submersible fluorometer designed for the continuous in situ recording of concentrations of materials in an aqueous environment, said materials being stimulated to fluoresce when excited by light of proper wavelengths.

United States Patent [191 Leaf 5] Aug. 21, 1973 IN SITU FLUOROMETER [56]References Cited [75] Inventor: William Benjamin Leaf, Silver UNITEDSTATES PATENTS P B 3,354,772 11/1967 Topol 250 218 250/71 R [731Assignees. Prototypes, Incorporated, 2'403'631 7/1946 Bmwn Kensington,Zone Research 3,151,204 9/1964 Stacy 250/71 R Incorporated, Washington,DC. Primary Examiner-James W. Lawrence [22] Fled: 1972 AssistantExaminer-T. N. Grigsby [21] APPL 224,713 Attorney-Sughrue, Rothwell &Mion Related US. Application Data [62] Division of Ser. No. 134,781,April 16, 1971, Pat. No. [57] ABSTRACT Disclosed herein is aself-contained submersible fluorometer designed for the continuous insitu recording Cl 250/7 R, 250/220 SD of concentrations of materials inan aqueous environ- [BL G0 G0 1 11 21/26, G0ln 2l/38 ment, saidmaterials being stimulated to fluoresce when [58] Field of Search250/218, 220 SP, ex ited by light of proper wavelengths.

1 Claim, 9 Drawing Figures Patented Aug. 21, 1973 3,754,145

2 Sheets-Sheet l FIGI Patented Aug. 21, 1973 3,754,145

2 Sheets Sheet 2 I0) I q REFERENCE- REF AMPLIFIER /30 LIGHTFROI- ANDLAIPZ DETECTOR WAVESHAPER I 6 LIGHT FROM 28) 34 3/ FILTERB PHOTOSYNCHRONOUS INTEGRATOR I AND we DETECTDR FIBER I6 DETECTOR I I IAUTOMATIC I GAIN com 3 ETQWKWOTEWTWETTT sAIPLE (a) IATERIALCONCENTRATION /STEP|NCREASE REFERENCE (b) AMPLIFIER- SHAPER OUTPUT LIGHTFROM FLUORESCENCE PHOTO OF SAMPLE MATERIAL T DETECTOR 28 P 4 4 LIGHTFROM OPTIC OUT UT FIBER BALANCE PATH-I6 (d) DETECTOR 34 A INPUT DETECTOR34 9 OUTPUT V \AA INTEGRATOR as f OUTPUT (DRIVES SERVO AMPLIFIER) INSITU FLUOROMETER This is a division of application, Ser. No. 134,781filed Apr. 16, 1971 and now US. Pat. No. 3,649,833.

BACKGROUND OF THE INVENTION The invention is in the field offluorometers and more specifically in the field of fluorometers used tomeasure concentrations of fluorescing material, said concentrationmeasurements being used to study the effects of pollutants and man'sactivities in the aqueous environment.

The basis of fluorometer measurements to detect material concentrationsis that certain materials exhibit unique, repeatable fluorescentcharacteristics. In particular, when excited by light of certainwavelengths, these materials respond by emitting light in another,longer wavelength band. By selecting proper light sources, opticalfilters and optical detectors, materials which exhibit fluorescence canbe detected and their concentrations measured with a great degree ofaccuracy over a wide range of concentrations in various naturalbackgrounds.

The detectability of the unique fluorescent signatures of variousmaterials in solution forms the basis for the application of thefluorometer to studies directed at the solution to water pollutionproblems. Fluorometer measurements are extremely useful in the area ofwater pollution studies from two aspects. First, by injecting samples ofa fluorescent tracer material into a body of water, studies may be madeof the dispersion and dilution rates of aqueous contaminant fields.Thus, fluorometer measurements can be used to predict the effect of theinjection of contaminant fields into the water body. Second, it ispossible to determine the effects of pollution or human activities onthe ability of a body of water to support astanding crop ofphytoplankton which is a basic link in the marine food chain. This isachieved by the measurement of fluorescent chlorophyll which is found inthe phytoplankton and forms a sensitive indicator of the presence anddensity of phytoplankton in the aqueous environment under study.

In the past, such studies have been attempted by pumping samples of thebody of water under consideration to surface fluorometers or by thecollection of discrete deep samples by submerged sampling bottles.However, there are many disadvantages with these prior methods. First,pumped samples become mixed or smeared in the pumping hose and theresolution and sensitivity of a surface fluorometer to detect smallscale concentration gradients is lost. Further, it is extremelydifficult to make continuous concentration profiles at depths greaterthan about meters. Finally, it has been impossible to access the changesin sample characteristics occasioned by pumping forces and by pressurechanges and temperature variations encountered by the sample duringtransit to the surface.

The above problems are overcome completely by the in situ fluorometer ofthis invention'which provides real-time, continuous measurementcapabilities. The insitu fluorometer operates within the aqueousenvironment itself thus eliminating the requirement of bringing a sampleof water to the surface. The fluorometer disclosed herein permitsconcentration measurements at depths of 200 meters and beyond,permitting dispersion and dilution studies at depths greater than 15meters where bottom effects are critical to understanding localdispersion characteristics.

Further, in the measurement and analysis of concentrations ofphotosynthetic phytoplankton a deep measurement capability isparticularly useful as the depth of the photic zone, in which solarenergy is utilized in the photosynthesis process, frequently exceeds 50meters and the phytoplankton drift or are mixed to considerably deeperdepths.

SUMMARY OF THE INVENTION The instrument of this invention provides aselfcontained submersible fluorometer for the continuous in siturecording of concentrations of fiuorescing material in a body of waterunder study. The fluorometer employs the double beam optical bridgeprincipal which permits the measurements of very low fluorescent levelswhich occur when measuring relatively small concentrations of materialsamples in large bodies of water.

Utilizing this principal, a light chopper permits a photodetector tolook alternately at the emission from the fluorescing sample and areference light in a standard balancing path' of the bridge. Thereference light in the balancing pathis adjusted by a servo driven,circular neutral density wedge filter which has a continuoustranmissibility variation of several decades of light. The photodetectoroutput is demodulated by a synchronous detector, using as asynchronousreference signal, light derived from the exciting light source choppedby the rotation of the light chopper. The detector output is used toenergize a servo motor. The polarity of the motor drive voltage is suchthat the circular neutral density wedge filter is rotated in thedirection required to maintain a light balance in the optical bridge.Coupled to the servo motor and the neutral density wedge filter is aposition potentiometer with a wiper arm geared to the neutral densityfilter. The output voltage from the position potentiometer is apotential proportionalto the intensity of the fluorescence which isindicative of the concentration of the sample material.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 illustrates the basic externalconfiguration of the fluorometer of this invention with a portion of theouter cover removed'to illustrate a section of the internal fluorometerstructure;

FIG.-2 illustrates the internal structure of the in situ fluorometer;

FIG. 3 is a schematic diagram of the electrical components of thefluorometer; and

FIGS. 4a thru 4f are a timing diagram corresponding to the operation ofthe circuitry of FIG. 3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT FIG. 1 illustrates thebasic configuration of the in situ fluorometer of the invention. Thefluorometer is generally cylindrical in shape having a faired nose piece9 defining an undulating internal passageway or duct 5 through whichwater is caused to flow. The instrument can either be towed behind amoving boat or placed at rest in a flowing stream with the faired noseportion of the instrument facing the current. In either case, thepressure difference between the intake duct 1 and the outlet duct 3causes water to flow through a transpar ent cuvette 6. Between the inlet1 and the outlet 3 is positioned a curved duct 5 for carrying the watersample to and from the cuvette 6. The duct 5 is internally blackened andis so shaped to block ambient light from reaching the cuvette.

Filter 4 passes light from an excitation source which emits radiation atwavelengths which excite the sample material, whose concentration is tobe detected, to fluorescence. As the material fluoresces, filter 8passes only those wavelengths of light which correspond to thecharacteristic fluorescence of the sample material.

Coupled to the instrument is cable 7 which may serve as a towing cable,an information carrying line for transferring to the surface thedetected concentration information and for carrying operating power froma surface power supply to the instrument. It is, of course, possible forthe concentration information to be recorded on board the fluorometer byincluding therein a suitable recorder. Further, the fluorometer can betotally self-contained by including a battery within the cylinder 11 foroperating the instrument. Utilization of a battery within the instrumentalleviates the requirement of transferring power from the surfacethrough the cable 7 to the fluorometer. The cable may be removablyattached to cylinder 11 by a water proof connector.

The double beam optical bridge of this invention and its operation willnow be described with reference to FIGS. 2, 3, and 4. As watercontaining the sample material under study passes through transparentcuvette 6, light from lamp 2 passes through excitation filter 4 excitingthe material to fluorescence. Lamp 2 may be a fluorescent lamp or anyother light source which emits radiation containing wavelengths suitablefor exciting the material under study. Excitation filter 4 permits thepassage of only those wavelengths suitable for exciting the material.The resulting fluorescent light as well as some light from filter 4impinge emission filter 8. Filter 8 is selected to pass only thosewavelengths from the desired fluorescent light to the photodetector 28.

The light chopper comprises a light shutter 24 rotated by shutter motor26. The underside ofthe shutter is reflective to indicent light. Thus,when the shutter is in the position shown in FIG. 2, the light outputfrom filter 8 is blocked from the photodetector 28, while the lightoutput from optic fiber 16 is reflected onto the photodector by theunderside of the shutter. Optic fiber I6 is positioned between the lightshutter 24 and the neutral density wedge filter 18 such that the inputto the optic fiber is controlled by the transmissibility of the wedgefilter. Thus, optic fiber 16 provides the balancing path, with the lighttraveling therethrough acting as the reference light for the double beamoptical bridge.

The light for the balancing pathes transmitted by optic fiber 14, oneend of which is positioned to receive light from lamp 2 with the otherend being positioned to emit light toward the underside of the wedgefilter opposite the lower end of the optic fiber 16. It should beunderstood that optic fiber 14 can be deleted by utilizing a secondlight source radiating directly on the underside of the wedge filter 18.

When the bridge is balanced, wedge filter 18 is positioned such that theintensity of light at the output of optic fiber 16 is equal to theintensity of the fluorescent light originating in cuvette 6 and passingthrough filter 8. The position potentiometer 22 is geared to the;

wedge filter so that its output potential uniquely defines the wedgefilter position. Since the position of the wedge filter is correlatibleto the intensity of the fluorescence, and since the amount offluorescence is proportional to the concentration of the sample materialin the water flowing through cuvette 6, the output potential of thepotentiometer is thus proportional to the sample concentration.

By continuously plotting the output of the potentiometer 22 as thefiuorometer is caused to move either vertically or horizonally throughthe body of water under study, vertical and horizonal profiles ofmaterial concentrations can be obtained for use in circulation anddilution studies. For example, to predict the dispersion of aqueouscontaminant fields in a body of water, a tracer dye such as rhodamine Bdye can be added to the aqueous environment. Filter 4 is then selectedto pass radiation which excites the rhodamine to fluorescence whilefilter 8 is selected to pass the wavelength band characteristic offluorescing rhodamine.

Since the dispersion of the tracer dye-will generally correspond to thedispersion of a contaminant injected into the water as industrial waste,an accurate prediction of the dispersion of such industrial waste withinthe body of water can be made.

On the other hand, if the concentration of phytoplankton is to bedetermined, one need only replace excitation filter 4 with a filterwhich passes radiation over those wavelengths which excite chlorophyllto fluorescence. Emission filter 8 is selected to pass those wavelengthscharacteristic of fluorescing chlorophyll. Photodetector 28 is selectedto be sensitive to all the wavelengths under consideration. That is, inmaking concentration measurements the photodetector is sensitive to thewavelengths emitted by the excitation lamp 2 as well as the wavelengthswhich pass through the emission filter 8. r

The operation of the light bridge will now be described with particularreference to FIGS. 3 and 4. Common numerical destinations in FIGS. 2 and3 identify common elements. Thus, photodetector 28 in H6. 2 correspondsto the like identified photodetector in FIG. 3.

As shutter 24 rotates under the control of motor 26 the input tophotodetector 28 alternately receives light from the optic fiber l6 andthe emission filter 8. The output of the photodetector is coupled to acontrol circuit 12. The control circuit is shown in detail in FIG. 3.The photodetector output is coupled to a preamplifier 31. Thus, theoutput from the photodetector is amplified and passed to a bandpassamplifier 32. The output from amplifier 32 appears as the input tosynchronous detector 34. A synchronous reference signal is derived froma reference photodetector 10 which receives light from the excitationlamp 2. The light which impinges reference detector 10 is chopped by thelight shutter. 24 as it rotates under the control of the shutter motor26. The synchronous detector output is coupled to an integrator 36 andan automatic gain control circuit 40. The automatic gain control circuitis connected in a feedback loop to the bandpass amplifier 32, and actsto maintain a constant reference or (1.0. level in the output of theamplifier. The output of the integrator is supplied to a differentialservo amplifier 38 which controls the rotation of servo motor 20. Servomotor 20 is mechanically coupled to the neutral density wedge filter 18and to the wiper arm of the position potentiometer 22. The potentiometerwiper arm is electrically coupled by line 25 to a recorder 42 whichrecords the potentiometer output potential.

Let it be assumed that the sample material concentration is initially atsome low level as illustrated in FIG. 4a. As shutter 24 rotates,chopping the input light to reference photodetector 10, a synchronizingsignal is generated and applied to the amplifier and wave shapercircuitry 30. The output of circuit 30 is the square wave illustrated inFIG. 4b. With the concentration of the sample material at a constantlevel for a time sufficient to balance the optical bridge, the output ofthe photodetector 28 appears as a generally constant level waveform(FIG. 4c). The output from the photodetector 28 passes through thebandpass filter-amplifier 32 to produce an input to the synchronousdetector 34 as illustrated in FIG. 4d. The output of the detectorappears as illustrated in FIG. 4e. The integrator acts to integrate theoutput from the detector 34 to produce a waveform as illustrated in FIG.4f. Synchronous detector circuits are generally known in the art and afull description thereof is unnecessary for an understanding of theinvention. They generally function to produce an output signalindicative of the mismatch or difference between the two portions of amixed input signal that has been demodulated in accordance with asynchronizing signal.

Let it now be assumed that there is a rapid rise in the concentration ofthe fluorescing sample material in the body of water through which thefluorometer is passing. This is illustrated in FIG. 4a by a stepincrease in the sample material concentration. Since an increase insample material concentration causes a corresponding increase in theintensity of fluorescence, there is a rapid rise in the output from thephotodetector 28 during those periods when it is viewing the light fromthe euvette, thus causing a fluctuating signal to appear at the input tothe detector 34 as shown in FIG. 4d.

This results in a pulse train at the output of detector 34, as shown inFIG. 4e, whose magnitude decreases as bridge balance is restored. As aresult, the output from the integrator begins to increase causing animbalance between the inputs to the differential servo amplifier 38. Theservo motor begins to rotate in a direction to drive the circularneutral density wedge filter 18 in a direction required to restore alight balance in the optical bridge. Thus, in this example, the servomotor would rotate in a direction to cause the transmissibility of thewedge filter 18 to increase in the area between the optic fibers 14 and16. In addition, as the servo motor rotates, the wiper arm of positionpotentiometer 2 moves in the direction to balance the inputs to thedifferential servo amplifier 38. Thus, as seen from FIG. 4c the outputfrom photodetector 28 corresponding to the light from the optic fiber 16increases in response to the detection of an increase in fluorescence ofthe sample material. As the intensity of light in the balancing pathincreases, the intensity difi'erence in the two arms of the bridgedecreases thus explaining the decaying waveform in FIG. 4d. As a result,there is a corresponding decrease in the output from detector 34.

As previously described, the automatic gain control circuit 40 assuresthat the output of the bandpass amplifier 32 assumes a constantreference level when the bridge is balanced. Integrator 36 may be aresistancecapacitor circuit which stores energy received from thedetector. When the output of the detector falls back to its referencelevel in response to the balancing of the optical bridge, the output ofthe integrator levels off at some new value determined by the 'energystored in the integrator circuitry.

The output potential from the position potentiometer 22 is recorded bycoupling the wiper arm of the potentiometer to a suitable recordingmeans 42 through cable 25. When the recording means is contained withinthe fluorometer, cable 25 runs directly to the recording means. However,if the recording means is located on the surface, cable 25 isincorporated into cable 7.

In an actual embodiment of the complete fluorometer of the invention,the cylindrical housing 11 may be provided with diving fins ordepressors, as is known in the art, to achieve a desired towing depthand stability when in use.

While the invention has particularly shown and described with referenceto a preferred embodiment thereof, it will be understood by thoseskilled in the art that various changes in form and detail may be madetherein without departing from the spirit and scope of the invention.

What is claimed is:

1. An apparatus for measuring in situ the light generated by fluorescingmaterials suspended in a liquid medium comprising:

a. a transparent, hollow sample chamber open at both ends for carryingthe liquid medium,

b. means for illuminating the sample chamber to cause fluorescence,

c. means for sensing the light generated by fluorescing material withinthe sample chamber,

d. said sensing means comprising an optical bridge having two opticalpaths, the sample chamber being disposed in one path and reference meansbeing disposed in the other path,

e. a sealed container internally mounting the illuminating means and thesensing means,

f. means mounting the sample chamber within the container,

g. said container including a faired nose portion containing curvedinlet and outlet ducts connected to the open ends of the sample chamberfor providing a path for the liquid medium through the apparatus, thecurvature of the ducts shielding ambient light from the sample chamber,and

h. opaque, light absorbing means surrounding the container for furthershielding ambient light from the sample chamber.

1. An apparatus for measuring in situ the light generated by fluorescingmaterials suspended in a liquid medium comprising: a. a transparent,hollow sample chamber open at both ends for carrying the liquid medium,b. means for illuminating the sample chamber to cause fluorescence, c.means for sensing the light generated by fluorescing material within thesample chamber, d. said sensing means comprising an optical bridgehaving two optical paths, the sample chamber being disposed in one pathand reference means being disposed in the other path, e. a sealedcontainer internally mounting the illuminating means and the sensingmeans, f. means mounting the sample chamber within the container, g.said container including a faired nose portion containing curved inletand outlet ducts connected to the open ends oF the sample chamber forproviding a path for the liquid medium through the apparatus, thecurvature of the ducts shielding ambient light from the sample chamber,and h. opaque, light absorbing means surrounding the container forfurther shielding ambient light from the sample chamber.